The invention concerns a method for applying reactive films containing solids to microporous membranes, membranes produced accordingly and diagnostic elements which contain them.
So-called carrier-bound tests are often used for the qualitative or quantitative analytical determination of constituents of fluids, in particular of body fluids such as blood. In these tests reagents and in particular specific detection reagents and auxiliary reagents are embedded or immobilized in appropriate layers of a solid carrier. These layers are referred to as detection elements. The liquid sample is brought into contact with these detection elements in order to determine the corresponding analyte. The reaction of liquid sample and the reagents that are present initially in a dry form and are redissolved by the sample usually results in a signal that can be detected optically or electrochemically when a target analyte is present and in particular a color change which can be analyzed visually or with the aid of an instrument usually by means of reflection photometry. Other detection methods are for example based on electrochemical methods and detect changes in charge, potential or current.
Since, in contrast to conventional laboratory tests, the detection reagents are initially present in a dry form, carrier-bound tests are often also referred to as “dry chemistry tests”.
Test elements or test carriers for dry chemistry tests are often in the form of test strips which essentially consist of an elongate support layer made of plastic material and detection elements mounted thereon as test fields. However, test carriers are also known which are designed as square or rectangular wafers.
The photometric detection of low molecular analytes inn blood by means of dry chemistry test strips usually comprises the separation of erythrocytes which interfere with the photometric measurement.
The enzymes required for the analyte detection are usually located in a water-resistant, insoluble film in which a hydrophobic matrix consisting of film formers contains all or at least some of the detection reagents (i.e., essentially enzymes and indicator system), into which the sample penetrates and in which the color-forming reaction takes place. These films are applied by means of various established coating methods (e.g., knife-coating) on non-absorbent, mechanically stable support materials (such as, e.g., Pokalon® foil made of bisphenol-A polycarbonate).
The term film former means polymers which allow mechanically stable, water-resistant reagent layers to be coated (e.g., Propiofan® a vinyl propionate plastic dispersion).
In addition, these reactive films usually contain swelling agents. Swelling agents are water-soluble polymers which substantially influence the viscosity of the coating paste, which result in a fine dispersion of the reagents in the hydrophobic partial zones of the water-resistant layer and which facilitate the penetration of the sample into the layer (examples are alginate, Keltrol®, Gantrez®, Eudragit, etc.)
The “open porosity” and thus the ability of the analyte to penetrate into the reactive film can be positively affected by the addition of fillers (also known as film openers) (cf., e.g., U.S. Pat. No. 4,312,834). Fillers are water-insoluble, non-swelling, readily wettable, fine, inorganic or organic particles which do not optically scatter light or only to a slight degree and enable even relatively large molecules (for example lipids in the form of lipoproteins) and even cells (e.g., erythrocytes) to penetrate into water-resistant films. Examples of fillers are chalk, cellulose, diatomaceous earth, Celatom, kieselguhr, silicic acid, etc.
In the first generation of blood glucose test strips (e.g., “Hämoglukotest” 20-800 from Boehringer Mannheim, cf., also U.S. Pat. No. 3,630,957) the reactive film only contained a film former (Propiofan®) and a swelling agent (alginate) in addition to the detection chemistry. In the case of these very dense i.e., less open-pored, wipe-resistant films erythrocytes cannot penetrate into the reactive film, although tow molecular weight constituents of the blood such as in particular glucose are indeed able to penetrate. Hence, a separate blood separation was not necessary. The drop of blood in which it was intended to determine blood glucose was simply applied directly onto the reactive film of the test strip. After one minute incubation of the blood drop on the reactive film, the blood was wiped off, after a further minute reaction time the color development could be read from the same side of the strip to which the blood was previously applied as a measure of the analyte concentration.
Hence, it was for the first time possible to detect glucose directly in whole blood. Since these reactive films contained no fillers, they only allow the slow penetration of low molecular weight, readily water-soluble analytes such as glucose but not the detection of large and hydrophobic molecules (such as, e.g., cholesterol (CHOL), HDL (high density lipoprotein, i.e., lipoproteins of higher density), triglycerides (TG), creatine kinase (CK), etc.).
The use of qlass fiber fleeces to separate erythrocytes (see among others U.S. Pat. No. 4,816,224) especially in combination with open-pored reactive films containing fillers (e.g., the test strips of the Reflotron product line from Roche Diagnostics and later the so-called “non-wipe tests” of the Accutrend line from Roche Diagnostics) was a milestone in the development of dry chemistry tests for detecting analytes in whole blood. In addition to considerably more rapid kinetics, especially with regard to the penetration of the analyte into the detection film, enzymatic reaction and color reaction, these test superstructures also enable the detection of relatively large, hydrophobic molecules (e.g., CHOL, HDL, TG, etc.).
However, a disadvantage of the glass fiber fleece technology is the relatively unfavorable ratio of the volume of usable plasma to the blood volume used (also referred to as blood/plasma yield in the following). Furthermore, the supply of oxygen to the reactive film proved to be a limitation in the case of an oxidative analyte detection (i.e., in an analyte detection using analyte oxidase and reaction of the hydrogen peroxide reformed with peroxidase in the presence of an indicator which is converted in this process from a (usually colorless) reduced form into an (usually colored) oxidized form) especially in so-called stacked structures (glass fiber fleece for separating the erythrocytes and the reactive film from a stacked composite; the blood sample is applied to the glass fiber fleece, it penetrates the glass fiber fleece while separating the red blood cells and the serum or plasma formed in this manner penetrates into the underlying reactive film layer where the actual detection and indicator reaction takes place which can then be observed from the side of the stacked composite that is opposite to the blood application site) so that it is only possible to achieve a measuring range that is limited at the top end.
Thus, in order to reduce the blood volume, the most recent generation of test strips uses blood-separating membranes (cf., e.g., European Patent No. A 0 654 659) or very thin one-layer or two-layer films (cf., U.S. Pat. Nos. 5,536,470 and 6,036,919). The blood/plasma yield of such membrane-based systems is usually considerably more advantageous than is the case with glass fiber technology. Both membrane-based systems are elucidated in the following.
U.S. Pat. No. 5,536,470 discloses test fields which consist of a thin film layer. A sample of whole blood is applied to one side of the film layer. A color reaction can be detected from the opposite side without the erythrocytes being able to penetrate from the sample application side to the detection side. The film layer can be coated on a transparent support (e.g., foil) or on a membrane. Hence, the film disclosed in U.S. Pat. No. 5,536,470 acts as a combined blood (colored substance) separation and detection layer. A high proportion of pigment is necessary to fulfill the former function (blood (colored substance) separation), i.e., the pigment content is at least 30% by weight in this case based on the solids content of the film-forming paste. A high content of film former is also necessary to ensure the mechanical stability of such film layers containing a high proportion of pigment. The pigment and film former should be present in approximately the same weight ratio. Inert fillers (i.e., so-called film openers) should if possible not be present in these film layers or, if they are present, then they should only be present in the film forming paste in very small amounts (less than 10% of the total solids content) because otherwise the blood-separating property of the film layer is no longer ensured. However, due to the low filler content in the film-forming paste of at most 10%, the films disclosed in U.S. Pat. No. 5,536,470 are not sufficiently open-pored to be permeable to large, hydrophobic analytes (e.g., lipids).
In the case of a glucose detection using thin two-layer films on transparent foil, the first layer (i.e., the layer which rests directly on the foil) is a reactive film which contains film formers, swelling agents and an optically transparent filler (e.g., Transpafill®, a sodium aluminum silicate from Degussa) in addition to the enzyme-indicator system. In analogy to a wet chemical photometer test, the transparent first layer forms quasi the cuvette in which the photometric analyte detection occurs. The second layer applied to the first layer contains a high proportion of a highly refractive pigment (e.g., titanium dioxide) while dispensing with film openers or fillers. Blood is applied directly to the second layer, the photometric detection takes place from the opposite side of the test strip through the transparent support foil in the first layer.
The optically opaque, less open-pored second layer fulfills in this case a double function. On the one hand, as a blood-separating film it prevents erythrocytes from penetrating into the reactive first layer, and on the other hand, it reflects the light falling through the first layer and prevents the red erythrocyte color from shining through to the detection side.
The advantage of such a system compared to erythrocyte separation by means of a glass fiber fleece is the lower sample volume that is required and the rapid kinetics when detecting low molecular analytes.
The disadvantages of this two-layer structure is that large hydrophobic molecules (e.g., lipoproteins, cholesterol, triglycerides, HDL, etc.) cannot diffuse through the blood-separating second layer and can thus not be detected in the first layer.
Hence, an alternative is to use blood-separating membranes. Blood-separating membranes (i.e., membranes generating plasma or serum from whole blood) are very asymmetric membranes (usually polyether or polyether sulfone, e.g., BTS-SP-300 from the Pall Co., PrimeCareX or SG from Spectral Diagnostics), i.e., membranes whose pore diameter is not uniform, but rather have an open-pored and a narrow-pored side. Blood is usually applied to the more open-pored side of the membranes. The erythrocytes are held back in the tapering pores as the sample material passes through the membrane (cf., European Patent No. 0 654 659).
Blood separating membranes are basically used in two forms in dry chemistry test strips. In the so-called one layer structure the blood-separating membrane in addition to blood separation also fulfills the function of a support for the detection chemistry. For this purpose the membrane is impregnated with a system comprising an aqueous indicator and detection system (e.g., by means of bath impregnation or slot nozzle metering).
In order to ensure a rapid dissolution of the impregnated and dried enzymes and a rapid wetting of the membrane by the sample material, wetting agents are usually added to the impregnation solution.
A disadvantage of the one-layer membrane structure is that the membrane is optically non-transparent in the dry state (the refractive index of air is about 1.00; the refractive index of the membrane is about 1.35-1.38, i.e., the difference between the refractive indices is about 0.35-0.38 so that the membrane appears to be non-transparent), however, it becomes optically considerably more transparent in the wet state (the refractive index of water is about 1.33 so that the difference between the refractive indices is only about 0.02-0.05) and thus the intrinsic color of blood of the erythrocytes separated in the lower membrane zones shines through and influences the photometric measurement.
This can be reduced or prevented by adding white pigments (e.g., titanium dioxide, refractive index about 2.55) to the impregnation solution. Since optically opaque white pigments have particle sizes in the range of half the wavelength of the light to be reflected (0.2 to 0.4 μm), they can enter the pores of the membrane (which typically have a diameter of 0.2 to 10 μm) during the impregnation and thus narrow and block them and hence make it impossible or more difficult for large hydrophobic molecules to enter and pass through the membrane.
Consequently, one-layer structures with blood-separating membranes are used exclusively to detect small, readily water-soluble analytes (e.g., glucose).
A two-layer membrane structure allows many problems of the one-layer structure to be circumvented. In this case another, more narrow-pored detection membrane which absorbs the plasma from the blood-separating membrane (e.g., Biodyne A or Loprodyne=0.2/0.45 μm nylon membrane from the Pall Company) is adjacent to the blood-separating membrane. In this case optically opaque white pigments are not necessary. Furthermore, the detection system present in the second membrane does not come into direct contact with the blood-separating system which, especially in the field of lipid tests, enables the use of wetting agents that readily dissolve lipids and also have a hemolytic effect.
However, disadvantages a the two-layer structure are a complicated, expensive test configuration. The manufacturing process makes high demands on the mechanical test strip assembly because a close contact without gaps if possible has to be ensured so that serum or plasma can pass from the blood-separating membrane into the detection membrane. Inherent disadvantages of the system are the slow wetting of the test structure, an unfavorable blood/plasma yield compared to the one-layer structure and slower kinetics due to the narrower pores of the detection membrane.
Thus, in summary the disadvantages of the methods of the prior art are that open detection films having a high proportion of fillers are necessary especially to detect large hydrophobic molecules, but such open detection films alone do not ensure a separation of interfering blood components (above all erythrocytes, hemoglobin) for test strips that are analyzed optically. In contrast, suitable blood separation systems (films, membranes) allow the penetration of large hydrophobic molecules, if at all, then only to an inadequate extent. Systems that are basically suitable for detecting large hydrophobic molecules (such as the combination of glass fiber fleece and an open detection film or two-layer membrane structures) only inadequately solve the problem because they are complicated to manufacture and prone to interference and are suboptimal in their test performance (large volumes of blood required, limited upper measuring range or slow reaction kinetics).